Not Applicable
The present invention relates to interfacing two-dimensional electrophoretic separations of biomolecules, such as protein, DNA, and carbohydrates involving capillary electrophoresis (CE). Specifically, the present invention relates to interfacing multiple capillaries either fabricated on a microchip or bundled together with multiple individual capillaries as in capillary array electrophoresis (CAE) with a different channel (capillary and/or strip gel) perpendicularly to provide sample transfer to multiple capillaries simultaneously.
Usually, electrophoresis separates protein mixtures based either on their charges or on their sizes (molecular weights). By combining these two mechanisms, which are orthogonal to each other, a particularly powerful tool called two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is formed (Kenrick, K. G. and Margolis, J. Isoelectric focusing and gradient gel electrophoresis: a two-dimensional technique, Anal. Biochem. 1970, 204-207; O""Farrell, P. H. High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem. 1975, 250, 4007-4021). The modern two-dimensional electrophoresis has improved significantly with many modifications to the original technique developed almost thirty years ago. However, the general procedures remain the same, typically involving sequential separations by first dimension of isoelectric focusing (IEF) and the second dimension of slab gel electrophoresis (SGE) using sodium dodecyl sulphate (SDS).
This 2-D PAGE technology is the only technique known so far capable of separating thousands of proteins simultaneously and providing highly purified proteins. Actually, 2-D PAGE is the core technology that forms the basis for the rapid expanding field of proteomics and genomics (Wilkins, M. R., Williams, K. L., Appel, R. D. and Hochstrasser, D. F., Eds, Proteome Research: New frontiers in Functional Genomics, Springer, Berlin, 1997). Currently, proteomics and genomics heavily rely on 2-D PAGE and related technologies to separate, identify and quantitate proteins. Unfortunately, the current 2-D PAGE technique requires separate steps and is very hard to automate. Therefore, it is critical for the future development of proteomics to automate the protein separation and quantitation process. To date, very few successful results have been reported for the automation of 2-D PAGE.
Recently, capillary electrophoresis (CE) has emerged as a powerful separations technique, with applicability toward a wide range of molecules from simple atomic ions to large DNA fragments. In particular, two of the operational modes, i.e. capillary IEF (cIEF) and capillary gel electrophoresis (CGE), have become attractive alternatives to slab gel electrophoresis for biomolecule analysis, including protein separation and DNA sequencing. This is generally attributed to the fact that the small size of the capillary greatly reduces Joule heating associated with the applied electrical potential. Furthermore, cIEF and CGE produce faster separation with better resolution than slab gels. Especially, the sub-nanoliter sample volume requirement make these technique extremely attractive for biomedical analysis where samples are often too hard to get enough for other techniques to work. Because of the sub-nanoliter size of the samples involved, however, a challenging problem in applying this technology is to handling the samples including transferring the samples from one dimension to another.
Isoelectric focusing separation of proteins in an immobilized pH gradient (IPG) is extensively described in the art. The concept of the immobilized pH gradient (IPG) is disclosed in U.S. Pat. No. 4,130,470 and is further described in numerous publications (Bjellqvist, B., Ek, K., Postel, W., Isoelectric focusing in immobilized pH gradients: principle, methodology, and some applications, J Biochem. Biophys. Methods 1982, 6,317-339; Gorg, A. Postel, W., Gunther, S., Weser, J., Improved horizontal two-dimensional electrophoreis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying-on transfer to the second dimension, Electrophoresis, 1985, 6,599-604).
It is current practice to create IPG gels in a thin planar configuration bonded to an inert plastic sheet that has been treated for chemical binding to an acrylamide gel. The IPG gel is typically formed as a rectangular plate of 0.5 mm thick, 10 to 30 cm long (in the direction of separation) and about 10 cm wide. Multiple samples can be applied to such a gel in parallel lanes, with the attendant problem of diffusion of proteins between lanes producing cross contamination. In the case where it is important that all applied protein in a given lane is recovered in that lane (as is typically the case in 2-D electrophoresis), it has proven necessary to split the gel into narrow strips, (Immobiline DryStrips, typically 3 mm wide), each of which can then be run as a separate gel. Since the protein of a sample is then confined to the volume of the gel represented by the single strip, it will all be recovered in that strip.
IEF can also be performed in capillaries (Hjerten, S., Zhu, M. D., Adaptation of the equipment for high-performance electrophoresis to isoelectric focusing, J. Chromatogr. 1985, 346, 265-70; Hjerten, S., Liao, J. L, Rapid separation of proteins by isoelectric focusing in the high-performance electrophoresis apparatus, Protides Biol. Fluid. 1986, 34, 727-30; Thormann, W., Tsai, A., Michaud, J. P., Mosher, R. A., Bier, M., J. Chromatogr. 1987, 389, 75-86). In cIEF, the pH gradient is usually provided by supplying the full capillary with a mixture of the heterogeneous ampholytes and the homogeneous separation medium along with protein samples. The current cIEF suffers from two major limitations. One is the detection of the separated proteins bands. Since the whole content is restricted within the capillary, a solution with either high salt concentration or extreme pH has to be used to elute the analytes out from one of the capillary end for detection. Recently, some work to image the whole capillary for the separated bands have been reported (Fang, X., Tragas, C., Wu, J., Mao, Q., Pawliszyn, J., Recent development in capillary electric focusing with whole column imaging detection, Electrophoresis, 1998, 19, 2290-2295). The second limitation is the difficult to create the equivalent of IPG strips in the capillary due to geometric restriction (Hochstrasser, D., Augsburger, V., Funk, M., Appel, R., Pellegrini, C., Muller, A. F., Immobilized pH gradients in capillary tubes and two-dimensional gel electrophoresis, Electrophoresis, 1986, 7, 505-11). Therefore, the whole buffer system migrates, due to electroosmotic flow, during the IEF process making the focusing process difficult to reproduce. Coating the capillary surfaces with a hydrophilic layer reduces the electroosmosis and thus the buffer migration process (Bao, J., Separation of proteins by capillary electrophoresis using an epoxy based hydrophilic coating, J. Liq. Chrom. and Rel. Technol., 2000, 23, 61-78). However, most of the coatings cannot resist the extreme pHs involved in the IEF process for long. Fortunately, most of the cIEF can be accomplished within a shorter period of time due to a much higher voltage in cIEF as compared with traditional IEF process. Therefore, cIEF is still a very practical technology for protein separations.
Further, cIEF can also be performed on microchips. The details of this art have been described in details in references (Dolnik, V., Liu, S., and Jovanovich, S., Capillary electrophoresis on microchip, Electrophoresis 2000, 21, 41-54).
The principle and practices of SDS-PAGE are also extensively described in the art. It is current practice to detect proteins in SDS-PAGE gels either by staining the gels or by exposing the gels to a radiosensitive film or plate (in the case of radioactively labeled proteins). Staining methods include dye-binding (e.g., Coomassie Brilliant Blue), silver stains (in which silver grains are formed in protein-containing zones), negative stains in which, for example, SDS is precipitated by Zn ions in regions where protein is absent, or the proteins may be fluorescently labeled. In each case, scanners can be used to acquire the spot pattern images of the separated proteins. The images can be reduced to provide positional and quantitative information on sample protein composition through the action of suitable computer software. All of these detection techniques have their limitations, especially in terms of quantitation. Better method is desirable.
SDS-PAGE can also be performed in capillaries. Capillary SDS-PAGE has demonstrated its power in protein separation (Cohen, A. S., Karger, B. L, J. Chromatogr. 1987, 387, 409) as well as in DNA separation (Yeung, E. S., Li, Q., DNA sequencing by multiplexed capillary electrophoresis, in High Performance Capillary Electrophoresis, edited by Morteza G. Khaledi, Chemical Analysis Series, Vol. 146, 1998, John Wiley and Sons, pp 767-89). Capillary SDS-PAGE has many unique advantages such as easiness for detection and quantitation. When used for protein separation, all known on-line detection techniques for capillary electrophoresis (CE), such as WV-Vis, LIF (laser induced fluorescence) and MS detection can be used for the quantitation of the proteins. This feature is critical for providing quantitative information about the proteins being separated. Therefore, it is advantageous to use capillary SDS-PAGE for final protein determination.
Both IEF and SDS-PAGE have played important roles in advancing modern biology and chemistry. However, pharmaceutical companies facing strong competition and regulatory pressure are designing more and more experiments, which result in large numbers of samples for evaluation. The conventional slab gel based electrophoresis and single capillary-based CE cannot meet the ever-demanding need for sample throughput. One solution is to conduct analysis of multiple samples simultaneously in many capillaries bundled together, i.e., multiplexed simultaneously order to same time and reduce cost. For example, attempts have been made to sequence DNA in slab gels with multiple lanes to achieve multiplexing. However, slab gels are not readily amenable to a high degree of multiplexing and automation. Difficulties exist in preparing uniform gels over a large area, maintaining reproducibility over different gels, and loading sample wells. Furthermore, difficulties arise as a result of the large physical size of the medium, which requires a uniform cooling, large amounts of media, buffer, and samples, and long run times for extended reading of nucleotides. However, capillary electrophoresis can be highly multiplexed and run in parallel. The substantial reduction of Joule heating per lane makes the overall cooling and electrical requirement more manageable. The cost of materials per lane is reduced because of the smaller sample sizes. Therefore, the advantages of CE can produce substantial gain in shortening the time needed for various sample assays.
Both IEF and SDS-PAGE have proven to be important separation techniques. Further, they can be combined together to form a two-dimensional electrophoresis (2-D PAGE) system and to give more separation power as they are orthogonal separation techniques, which means that the separation parameter for SDS-PAGE, mass, is almost completely unrelated to the separation parameter of IEF (pI). The theoretical resolution of the 2-D system is the product of the resolutions of each of the constituent methods, which is in the range of 150 molecular species for both IEF and SDS electrophoresis. This gives a theoretical resolution for the complete system of 22,500 proteins, which accounts for the intense interest in this method. In practice, 5,000 proteins have been successfully resolved experimentally.
Two-dimensional electrophoresis is widely used to separate from hundreds to thousands of proteins in a single analysis, in order to visualize and quantitate the protein composition of biological samples such as blood plasma, tissues, cultured cells, etc. The technique was introduced in 1975 by O""Farrell, and has been used since then in various forms in many laboratories (O""Farrell, P. H., High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem. 1975, 250, 4007-4021; Klose, J., Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues; A navel approach to testing for induced point mutation in mammals, Humangenetik, 1975, 26, 231-243; Scheele, G. A., Two-dimensional gel analysis of soluble proteins: characterization of guinea pig exocrine pancreatic proteins, J. Biol. Chem. 1975, 250, 5375-5385). Even today, 2-D PAGE is still the only known technique capable of separating thousands of proteins in complex samples. (Herbert, B. R., Sanchez, J. C. and Bini, L., Two-dimensional electrophoresis: the state of the art and future directions, in Proteome Research: new frontiers in functional genomics, Wilkins, M. R., Williams, K. L., Appel, R. D., and Hochstrasser, D. F. Eds. pp13-34, 1997). Because 2D-PAGE has the ability to provide detailed views of thousands of proteins expressed organism or cell type, it has undeniably assumed a major role in protein separation and identification (Cash, P., Anal. Chim. Acta, 1998, 372, 121-145).
The key to the success of 2-D PAGE is to have a proper way of transferring proteins separated by IEF into the second dimension. The transfer of biopolymers, such as proteins, DNA and RNA from free solution is relatively straightforward. But, transferring biopolymers from gels is problematic due to the physical barriers that the large molecules encounter. Usually, the biopolymers are well enmeshed inside the gel and can be released only after being coaxed strongly. Several methods have been used for recovering biopolymers from a gel. These methods include direct extraction, sonication, electroblotting, electroelution, and electrophoresis.
4. Extraction method is to directly cut out the gel with the band of interest, mash it and immerse it in a buffer solution containing SDS, glycine and Tris((tris-hydroxymethyl)-aminomethane). After shaking the mixture and filtering out the gels, the protein is recovered by extraction. This method is highly unsatisfactory for large proteins because large proteins diffuse very slowly within the complex gel network.
5. Sonication increases the rate of protein diffusion from within the pores of the gel out into the solvent. But, the efficiency is still low and the process takes a long time. In addition, excessive sonication may result in the degradation of the sample molecules.
6. Electroblotting, the most common and satisfactory method of recovering proteins, involves transfer of proteins from the gel onto another equally sized nitrocellulose membrane, using an electric current to drive their migration in a manner similar to the original electrophoresis, but in a perpendicular direction (Western blot). The reason for performing an electroblot is that the proteins are now more accessible on the transfer membrane than they were in the gel. For instance, detection techniques are more sensitive and the proteins on the membrane can be reacted in situ, with antibodies or other agents.
7. Electroelution is another commonly used method of recovering sample from gel. In this method, gel containing the nucleic acid of interest is cut out and put into a dialysis bag filled with buffer. After the gel has sunk to the bottom of the bag, the excess buffer is removed. The bag is then immersed in a shallow tank of buffer and electric current is passed through the bag. The nucleic acid is then electroeluted onto the wall of the bag. The polarity of the current is reversed for a short time to release the nucleic acid from the wall of the bag. The nucleic acid is thereby recovered and purified.
8. Electrophoresis is the best method for recovering samples from the gel. In a 2-D PAGE, proteins from the IEF gel is easily transferred to the SDS-PAGE gel by electrophoresis. After the IEF step, the IEF gel strip containing proteins separated by IEF is taken out of the IEF chamber and placed along the edge of the SDS-PAGE gel with the SDS-PAGE process in a direction perpendicular to the prior IEF separation. Protein molecules are transferred to the SDS-PAGE gel during the electrophoresis process to yield a two-dimensional (2-D) separation. This interface is the best approach available so far to transfer protein molecules from the IEF step to the SDS-PAGE step because it provides a seamless transition of proteins from the IEF gel to the SDS-PAGE gel. This is no surprise as electrophoresis is the most effective means of moving biopolymers in gel. The only drawback of this interface is that manual operation is required to take the IPG strip out and place it along the SDS-PAGE gel in order to complete the process. This manual procedure makes it difficult for automation of the whole process.
Moreover, the current IPG and slab gel systems are not fully automated, wherein all operations including gel casting, processing, sample loading, running and final disposition are carried out by an integrated, fully automated system. Current gel systems cannot be fully controlled by a computer and cannot systematically vary gel, process, sample load and run parameters, provide positive sample identification, and cannot collect process data with the object of optimizing the reproducibility and resolution of the protein separations. The current invention takes advantage of the electrophoresis principle and used capillary electrophoresis as a means of transferring the samples electrically into a capillary for convenient detection and analysis.
Several references in the literature describe the coupling of slab gel electrophoresis to mass spectrometry for protein characterizations (Busch, K. L, Interface device and process to couple planar electrophoresis with spectroscopic methods of detection, U.S. Pat. No. 5,245,185, Sep. 14, 1993.). This is a fundamentally different form of coupling (electrophoresis to mass coupling) and is not considered relevant to the present invention (electrophoresis to electrophoresis).
Capillary IEF can also be interfaced with different techniques through its outlet end. For example, MS has been used to interface with cIEF technique for protein analysis (Tang, Q., Harrata, A. K., Lee, C. S., Capillary isoelectric focusing-electrospray mass spectrometry for protein analysis, Anal. Chem. 1995, 67, 3515-19). In addition, there have been many reports attempting to interface HPLC with other electrophoretic techniques (Hanash, S. M.; Strahler, J. R. Advances in two-dimensional electrophoresis, Nature (London), 1989, 337, 485-6; Snider, J., Neville, C., Yuan, L. C., Bullock, J., Characterization of the heterogeneity of polyethylene glycol-modified superoxide dismutase by chromatographic and electrophoretic techniques, J. Chromatogr. 1992, 599, 141-55; Hooker, T. F., Jeffery, D. J., and Jorgenson, J. W., Two-dimensional separations in high-performance capillary electrophoresis, in High Performance Capillary Electrophoresis, edited by Morteza G. Khaledi, Chemical Analysis Series, Vol. 146, 1998, John Wiley and Sons, pp 581-612; Jorgenson, J. W. and Lemmo, A. V., U.S. Pat. No. 5,496,460). Further, cIEF has even been interfaced with traditional gel electrophoresis (Hirota, M., Development of a new type of two-dimensional electrophoresis and its application to the analysis of alkaline phosphatase isoenzymes in sera of pregnant woman, Yamaguchi Igaku 1986, 35, 87-95). However, this kind of interface still requires the move of proteins out of one end of the capillary, which is different from the current invention. Therefore, there has been no report on interfacing cIEF with multiplexed capillary array electrophoresis process. Especially, there is no report for interfacing cIEF perpendicularly with other techniques including SDS-PAGE. Further, there is no report for interfacing multiplexed capillary with another capillary array systems.
The present invention is aimed primarily at providing a means and a system for interfacing the multiplexed capillaries to different channels (or capillaries or gel strips) for the 2-D applications, and providing means for automating the whole 2-D process to afford higher throughput, resolution, speed, and automation.
It is an object of the present invention to provide a system for use in a 2-D system using all known methodologies associated with single capillary cIEF including sampling, detection, and elution.
It is a further object of the present invention to provide a cIEF system using all known methodologies associated with ultra thin EPG strips for cIEF.
It is a further object of the present invention to provide a multiplexed capillary array system for SDS-PAGE as second dimension in a 2-D process utilizing all known methodologies associated with single capillary SDS-PAGE including sampling, run parameters, detection and buffer selection.
The present invention provides an apparatus and a means for interfacing multiplexed CE capillaries with a channel that is used for conducting another dimension of separation. The apparatus is consisted of a channel, a substrate, a cover, and an interface.
A channel according to the present invention includes an opening disposed in a substantially planar substrate with a proper cover and at least one end opening of the channel is in fluid communication with external fluid through either a hole on the cover or the side surface of the substrate. If the channel openings extend through the side surface of the substrate, the channel opening can be manufactured simultaneously with the channel in the planar surface of the substrate. Thus, there is no need for specially drilled ports that must be aligned with the tiny channels from the sides. In this case, the channel should be fabricated to the edge of the substrate for connection with external capillaries. If the end opening is extended toward the reagent reservoirs on the outer surface, the whole channel is fabricated within the substrate and fluid communication is achieved through the hole on the cover. Preferred channel embodiments include at least a second channel opening that is coplanar with the first channel opening. The channel is suitable for transporting a sample during CE process.
Each channel can include a network of interconnected channels, if necessary, and each channel can include an opening that extends through one of the side surfaces of the substrate or an alternate surface of the substrate. For example, a plurality of channel openings can extend through the same surface to facilitate interaction with the interface as described below. Additionally, groups of channel openings can be spaced apart with certain distance on the substrate.
A substrate is used to house the channel. Various substrates ranging from glass to plastics can be used as the substrate. Channels can be made using different techniques available for different substrates. For example, molding or ablation can be used to fabricate channels on plastics. If silicone rubber is used as the substrate, a simple casting can be done with on a proper mold to generate the channels.
A cover can be made of different materials including glass and rubber and is used to cover the surface of the substrate with channels fabricated on it. If glass is used, there is only one way, i.e. from the side surface between the cover and the substrate, for interfacing capillary array with the channel. For rubber cover, in addition to the side surface, the interface can be accomplished from the front surface. This cover serves multiple purposes. First, it covers the channels fabricated on the substrate to form a capillary, through which CE process can be performed. Second, holes can be drilled on it to serve as reagent reservoirs to supply fluid to the capillary on the substrate. Third, electrodes can be placed in the reservoirs to provide electric contact for the capillary. Fourth, if the interface with capillary array is conducted through the outer surface (for rubber cover only), this cover sheet serves as an interface for capillary needles to insert in.
An interface having features of the present invention is useful for interfacing a channel having a pair of spaced apart channel end openings with externally applied fluids. In alternative embodiments the interface includes a capillary area, a conductor area, and an ionic fluid disposed in reagent reservoirs for establishing an electrical path between the channel area and the electrode. The ionic fluid is disposed in at least a portion of each end to establish the electrical path between the channel area and the conductor area. The ionic fluid can be the sample being tested or an alternate fluid, such as a separation buffer or other reactant.
The invention also includes a method for making a channel and a method for interfacing a channel opening with an electrical conductor and reactant fluids. Wherein the first dimension of separation, either electrophoretic (i.e., IEF or zone electrophoresis) or chromatographic, can be performed without interruption while the second dimension of CE separation can be run independently.
This multiplexing approach involves inserting a bundle of capillaries that are coupled individually in a capillary array or are fabricated in a microchip. The coupling can be done by inserting at least one capillary of the capillary array into the first dimension of separation channel that contains sample. It can also be accomplished by placing the tips of the capillary array adjacent to and perpendicular in relation to the first separation channel and apply electrical field to transport samples separated in first dimension to the second dimension.
The technique can be used for interfacing as many capillaries as desired, from at least 1 to ore than 1000 capillaries. The multiplexed capillary array electrophoresis (CAE) system contains an array of at least one (but possibly thousands) of capillaries, each preferably having an inside diameter of about 20-500 microns and a suitable outside diameter for the second dimension. Each capillary has an annular wall, an intake end, and an exit end. A more preferable inside diameter of each capillary is about 40-100 microns. The multiplexed CAE system may contain metal-coated capillary tips or metal tips glued to the capillary for better electric contact and better transfer of the samples from first dimension to the capillaries in the second dimension in CAE.
The present invention can be implemented utilizing an array of capillaries containing preferably at least about 1 capillary, and more preferably at least about 100 capillaries, and most preferably at least about 500 capillaries. A proper detector can be used to substantially monitor analyte separations by detecting the signals in a plurality of separation capillaries simultaneously. Inorganic materials such as quartz, glass, fused silica, and-organic materials such as Teflon(trademark) and its related materials, polyfluoroethylene, aramide, nylon, polyvinyl chloride, polyvinyl fluoride, polystyrene, polyethylene and the like can be used. Proper seal is preferably placed between the capillaries to avoid buffer leakage.
This multiplexed CAE system can be used for analyzing macromolecules such as proteins, amino acids, polypeptides, carbohydrates, polysaccharides, oligo-nucleotides, nucleic acids, RNAs, DNAs, bacteria, viruses, chromosomes, genes, organelles, fragments, and combinations thereof. This invention is also equally applicable whether a gel is used in the CE system or not.
A method for transferring macromolecules, such as biological molecules, between two different electrophoresis steps using a multiplexing approach is also provided. According to this method, samples are introduced into capillaries of a capillary array in a CE system.
A first aspect of the present invention is a novel method for interfacing multi-dimensional capillary electrophoresis. The novelty of this interface lies in several areas. 1. This is the first system offering an interface between a capillary channel (with or without gel) electrophoresis to another capillary electrophoresis. 2. The two electrophoreses are interfaced perpendicularly. 3. The perpendicular interface allows selective transfer of samples separated in the first dimension to the second dimension. This selectivity allows sampling specific sample zone without waiting for all prior samples to be eluted first, as is in a series transfer from the end of the channel. This feature is especially important in saving the time when the electrophoresis is connected to other fast response detectors, such as MS. 4. This interface is the first in allowing the simultaneous interfacing of multiple capillaries in the second dimension to the channels in the first dimension. 5. This interface also allows the multiple capillaries fabricated on a microchip to be interfaced with the first dimension channels.
The second aspect of the present invention is to separate molecules in channels or capillaries for both dimensions simultaneously. This feature allows the automation of the whole process.
The foregoing and other objects and aspects of the present invention are explained in detail in the drawings herein and the specification set forth below.